Division of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University, Gwangju 500-757, Korea.
Insect Biochem Mol Biol. 2011 Jan;41(1):42-50. doi: 10.1016/j.ibmb.2010.09.011. Epub 2010 Oct 27.
A bioinformatics search of the genome of the red flour beetle, Tribolium castaneum, resulted in the identification of two genes encoding proteins closely related to UDP-N-acetylglucosamine pyrophosphorylases (UAPs), which provide the activated precursor, UDP-N-acetylglucosamine, for the synthesis of chitin, glycoproteins and glycosylphosphoinositide (GPI) anchors of some membrane proteins as well as for the modification of other substrates. This is in contrast to other arthropods whose genomes have been completely sequenced, all of which have only a single copy of this gene. The two T. castaneum UAP genes, TcUAP1 and TcUAP2, share both nucleotide and amino acid sequence identities of about 60%. RT-PCR analysis revealed that the two genes differ in their developmental and tissue-specific patterns of expression. RNA interference (RNAi) indicated roles for TcUAP1 and TcUAP2 at the molt and intermolt stages, respectively: RNAi for TcUAP1 resulted in specific arrest at the larval-larval, larval-pupal or pupal-adult molts, depending on time of injection of double-stranded RNAs, whereas RNAi for TcUAP2 prevented larval growth or resulted in pupal paralysis. Analysis of elytral cuticle indicated loss of structural integrity and chitin staining after RNAi for TcUAP1, but not after RNAi for TcUAP2. Loss of peritrophic matrix (PM)-associated chitin was also observed following RNAi for TcUAP1, but not after RNAi for TcUAP2. Down-regulation of transcripts for either TcUAP gene at the mature adult stage resulted in cessation of oviposition in females, as well as fat body depletion and eventual death in both sexes. These results demonstrate that both TcUAP genes are critical for beetle development and survival, but that only TcUAP1 is clearly associated with synthesis of cuticular or PM chitin. However, both of these genes appear to have additional critical role(s) unrelated to chitin synthesis, presumably in the glycosylation of proteins and/or secondary metabolites.
利用生物信息学方法对赤拟谷盗（Tribolium castaneum）基因组进行搜索，发现了两个编码与 UDP-N-乙酰氨基葡萄糖焦磷酸化酶（UAPs）密切相关的蛋白的基因，这些酶为几丁质、糖蛋白和某些膜蛋白的糖基磷酸肌醇（GPI）锚的合成以及其他底物的修饰提供了活性前体 UDP-N-乙酰氨基葡萄糖。这与其他已完全测序的节肢动物形成了对比，这些动物的基因组中只有这一基因的一个拷贝。赤拟谷盗的这两个 UAP 基因，TcUAP1 和 TcUAP2，在核苷酸和氨基酸序列上具有约 60%的同一性。RT-PCR 分析显示，这两个基因在其发育和组织特异性表达模式上存在差异。RNA 干扰（RNAi）表明 TcUAP1 和 TcUAP2 分别在蜕皮和间皮阶段发挥作用：对 TcUAP1 的 RNAi 导致幼虫-幼虫、幼虫-蛹或蛹-成虫蜕皮时出现特异性停滞，这取决于双链 RNA 注射的时间，而对 TcUAP2 的 RNAi 则阻止幼虫生长或导致蛹瘫痪。对鞘翅角质层的分析表明，对 TcUAP1 的 RNAi 后结构完整性和几丁质染色丢失，但对 TcUAP2 的 RNAi 后则没有。对 TcUAP1 的 RNAi 后也观察到与围食膜（PM）相关的几丁质的丧失，但对 TcUAP2 的 RNAi 后则没有。在成熟成虫阶段下调任一 TcUAP 基因的转录物，导致雌虫停止产卵，以及两性脂肪体耗竭和最终死亡。这些结果表明，这两个 TcUAP 基因对甲虫的发育和生存都是至关重要的，但只有 TcUAP1 显然与角质层或 PM 几丁质的合成有关。然而，这两个基因似乎都具有与几丁质合成无关的其他关键作用（s），可能涉及蛋白质和/或次生代谢物的糖基化。
